Figure 2.

Overview of random library construction and selection for ODCase function. A: Codon randomization method. To eliminate any wild-type background, a StuI restriction site was first inserted at each target codon in pTP304. The StuI insert clones were then used as template for randomization of each codon by oligonucleotide directed mutagenesis. B: The random library in pTP304 was transformed into E. coli DB6656 and functional clones were selected on M9 minimal medium. The functional mutants were sequenced and results from the Lys73X library are shown. It is apparent that a positively charged residue is required at position 73 for ODCase function. Note that both of the possible lysine codons are included among the functional clones.